Molecular detection of and species and morphological characteristics of species in Japanese wild boars.

We investigated intraerythrocytic parasites in 21 Japanese wild boars, , captured in Wakayama Prefecture on the mainland from 2008 to 2009 and in 31 Japanese wild boars from 2011 to 2013 in Kochi Prefecture on Shikoku Island, Japan. We detected small subunit ribosomal RNA (18S rRNA) gene (SSUrDNA) fragments of a species in 17 boars from Wakayama and 18 boars from Kochi. The nearly full SSUrDNA sequence (1669 bps) of this species was determined.

Required Elements in tRNA for Methylation by the Eukaryotic tRNA (Guanine--) Methyltransferase (Trm11-Trm112 Complex).

The Trm11 and Trm112 complex (Trm11-Trm112) methylates the 2-amino group of guanosine at position 10 in tRNA and forms -methylguanosine. To determine the elements required in tRNA for methylation by Trm11-Trm112, we prepared 60 tRNA transcript variants and tested them for methylation by Trm11-Trm112. The results show that the precursor tRNA is not a substrate for Trm11-Trm112.

[Introduction of Team-based Learning to Evidence-based Medicine Educational Course for Pharmacy Students].

In Japanese pharmaceutical education, the Model Core Curriculum was revised in 2013 to train pharmacists who can appropriately evaluate literature and use evidence-based medicine (EBM). However, in the investigation of EBM education at pharmaceutical universities in 2015, it was found that literature evaluation was hardly performed in the education of undergraduate students. One of the reason is the lack of EBM lecturers at each universities.

[Evaluation of an Evidence-based Medicine Educational Program for Pharmacists and Pharmacy Students].

　Practicing evidence-based medicine (EBM) is likely to gain importance for clinical pharmacists in the relatively near future in Japan. An educational program including research and the critical appraisal of literature was required for pharmacy students as of 2015. We organized a six-month practical EBM course for pharmacy students at Hyogo University of Health Sciences.

[Evaluation of an Evidence-based Medicine Educational Program for Pharmacists and Pharmacy Students].

This study evaluated the effect of an evidence-based medicine (EBM) educational program on EBM-related knowledge and skills of pharmacists and pharmacy students. Our preliminary educational program included the following four sessions: 1) ice breaker, 2) formulation of answerable clinical questions from virtual clinical scenario using the PICO criteria, 3) critical appraisal of the literature using a checklist, and 4) critical appraisal of the results and integrating the evidence with experience and patients values. Change in knowledge and skills related to EBM were evaluated using pre- and post-seminar 4-point scale questionnaires comprising of 14 questions.

Continuous in vivo culture and indirect fluorescent antibody test for zoonotic protozoa of Babesia microti.

Babesiosis has attracted attention as a zoonotic disease. The disease is caused in immunocompetent individuals almost solely by Babesia microti, a rodent babesia. Most cases of human babesiosis by B.

Development of absolute quantification method for genotype-specific Babesia microti using real-time PCR and practical experimental tips of real-time PCR.

Babesia microti, a rodent babesia, is known as a pathogen of zoonosis, human babesiosis, is composed of several genotypes of small subunit ribosomal RNA gene (SSUrDNA) and different genotypes have been suggested to have different infectivity and pathogenicity to humans. We established a real-time PCR assay using SYBR Green I, which allows specific detection and absolute quantification for each SSUrDNA-type-B. microti of four SSUrDNA-types found in Japanese rodents even in mixed infection.

Development of real-time PCR assay for differential detection and quantification for multiple Babesia microti-genotypes.

We have developed a real-time PCR assay that can rapidly and differentially detect and quantify four genotypes of small subunit ribosomal RNA gene (SSUrDNA) of Babesia microti (Kobe-, Otsu-, Nagano- and US-types). In this assay, four genotype-specific pairs of primers targeted on internal transcribed spacer (ITS) 1 or 2 sequences were used and amplicons by each pair of primers were quantitatively detected by fluorescent SYBR Green I. The four genotype-specific pairs of primers displayed the high specificity for homologous genotype DNA.

The neurotrophic effects of glial cell line-derived neurotrophic factor on spinal motoneurons are restricted to fusimotor subtypes.

Glial cell line-derived neurotrophic factor (GDNF) regulates multiple aspects of spinal motoneuron (MN) development, including gene expression, target selection, survival, and synapse elimination, and mice lacking either GDNF or its receptors GDNF family receptor alpha1 (GFRalpha1) and Ret exhibit a 25% reduction of lumbar MNs at postnatal day 0 (P0). Whether this loss reflects a generic trophic role for GDNF and thus a reduction of all MN subpopulations, or a more restricted role affecting only specific MN subpopulations, such as those innervating individual muscles, remains unclear. We therefore examined MN number and innervation in mice in which Ret, GFRalpha1, or GDNF was deleted and replaced by reporter alleles.

Immunogold electron microscopic demonstration of distinct submembranous localization of the activated gammaPKC depending on the stimulation.

We examined the precise intracellular translocation of gamma subtype of protein kinase C (gammaPKC) after various extracellular stimuli using confocal laser-scanning fluorescent microscopy (CLSM) and immunogold electron microscopy. By CLSM, treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a slow and irreversible accumulation of green fluorescent protein (GFP)-tagged gammaPKC (gammaPKC-GFP) on the plasma membrane. In contrast, treatment with Ca(2+) ionophore and activation of purinergic or NMDA receptors induced a rapid and transient membrane translocation of gammaPKC-GFP.

Spatiotemporal analysis of the molecular interaction between PICK1 and PKC.

PICK1 is a protein which was initially identified as a protein kinase Calpha (alphaPKC) binding protein using the yeast two-hybrid system. In addition to alphaPKC, the PICK1 complex binds to and regulates various transmembrane proteins including receptors and transporters. However, it has not been clarified when and where PICK1 binds to alphaPKC.

Fused protein of deltaPKC activation loop and PDK1-interacting fragment (deltaAL-PIF) functions as a pseudosubstrate and an inhibitory molecule for PDK1 when expressed in cells.

To elucidate the role of 3-phosphoinositide-dependent protein kinase-1 (PDK1) in cellular signaling, we constructed and expressed a pseudosubstrate of PDK1, designated as deltaAL-PIF, and characterized its properties in cultured cells. deltaAL-PIF consists of two fused proteins of the protein kinase Cdelta (deltaPKC) activation loop (deltaAL) and PDK1-interacting fragment (PIF). The phosphorylation of deltaAL-PIF was detected with anti-deltaPKC phospho-Thr505-specific antibody and was increased in proportion to the expression level of co-expressed GST-PDK1, indicating that it acts as a pseudosubstrate of PDK1.

Propagation of gammaPKC translocation along the dendrites of Purkinje cell in gammaPKC-GFP transgenic mice.

To elucidate spatial and temporal profiles of the protein kinase C (PKC) activation in relation to neuronal functions including synaptic plasticity, we tried to detect PKC translocation in living brain slices. We first developed brain region-specific and inducible gammaPKC-GFP transgenic mice using a tetracycline (tet)-regulated system. In the transgenic mice, the expression of gammaPKC-GFP was region-specifically regulated by the promoter and abolished by the administration of doxycycline.